Methods for simultaneous amplification of target loci
The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.
1. A method of preparing a nucleic acid sample, the method comprising:
contacting the nucleic acid sample comprising at least 50 target loci with a library of at least 50 different primers to produce a reaction mixture;
subjecting the reaction mixture to PCR amplification reaction conditions to produce amplified products comprising target amplicons, wherein for at least 90% of the primers in the library the ΔG values for each possible combination of two primers are all equal to or greater than −10 kcal/mol, and wherein the at least 50 of the target loci are simultaneously amplified in a single reaction mixture; and
adding sequencing-compatible adaptors to the amplified products for sequencing by high-throughput next-generation sequencing.
2. The method of claim 1 , wherein for the at least 95% of the primers in the library the ΔG values for each possible combination of two primers in the library are all equal to or greater than −10 kcal/mol.
3. The method of claim 1 , wherein the target loci are SNP loci.
4. The method of claim 1 , wherein annealing temperature of the PCR amplification reaction is at least 3° C. greater than melting temperature of the primers.
5. The method of claim 1 , wherein annealing temperature of the PCR amplification reaction is at least 3° C. greater than highest melting temperature of the primers.
6. The method of claim 1 , wherein annealing temperature of the PCR amplification reaction is at least 8° C. greater than highest melting temperature of the primers.
7. The method of claim 1 , wherein annealing temperature of the PCR amplification reaction is at least 3° C. greater than average melting temperature of the primers.
8. The method of claim 1 , wherein annealing temperature of the PCR amplification reaction is at least 8° C. greater than average melting temperature of the primers.
9. The method of claim 1 , wherein range of melting temperatures of the primers is less than 5° C.
10. The method of claim 1 , wherein at least 100 target loci are amplified in a single reaction mixture.
11. The method of claim 1 , wherein at least 200 target loci are amplified in a single reaction mixture.
12. The method of claim 1 , wherein at least 90% of the amplified products are target amplicons.
13. The method of claim 1 , wherein at least 90% of the target loci are amplified.
14. The method of claim 1 , wherein less than 20% of the amplified products are primer dimers.
15. The method of claim 1 , wherein concentration of each primer in the library is less than 20 nM.
16. The method of claim 1 , wherein the primers have 2, 1, or 0 guanines or cytosines in the last 5 bases at the 3′ end of the primers.
17. The method of claim 1 , wherein the length of the annealing step of the reaction conditions is 3-60 minutes.
18. The method of claim 1 , wherein the target loci are SNP loci, and wherein the nucleic acid sample comprises cell-free DNA of mixed origin.
19. The method of claim 18 , wherein the nucleic acid sample comprises cell-free DNA from a transplant.
20. The method of claim 18 , wherein the nucleic acid sample is from a transplant recipient.
21. The method of claim 2 , wherein for at least 90% of the primers in the library the ΔG values for each possible combination of two primers in the library are all equal to or greater than −7.5 kcal/mol.
22. The method of claim 21 , wherein for at least 95% of the primers in the library the ΔG values for each possible combination of two primers in the library are all equal to or greater than −7.5 kcal/mol.
23. The method of claim 22 , wherein for at least 90% of the primers in the library the ΔG values for each possible combination of two primers in the library are all equal to or greater than −5 kcal/mol.
24. The method of claim 23 , wherein for at least 80% of the primers in the library the ΔG values for each possible combination of two primers in the library are all equal to or greater than −3.86 kcal/mol.
25. The method of claim 24 , wherein for at least 90% of the primers in the library the ΔG values for each possible combination of two primers in the library are all equal to or greater than −3.86 kcal/mol.